CN102796692B

CN102796692B - Method for improving sugar utilization rate of clostridium acetobutylicum in fermentation of mixed sugar

Info

Publication number: CN102796692B
Application number: CN201210163123.8A
Authority: CN
Inventors: 顾阳; 肖晗; 姜卫红; 宁媛媛; 李治林; 蒋宇; 孙喆; 杨晟
Original assignee: Shanghai Institutes for Biological Sciences SIBS of CAS
Current assignee: Nanjing Shiqi Biochemical Technology Co ltd
Priority date: 2011-05-25
Filing date: 2012-05-23
Publication date: 2014-12-17
Anticipated expiration: 2032-05-23
Also published as: CN102796692A; WO2012159571A1

Abstract

The invention discloses a method for improving the sugar utilization rate of clostridium acetobutylicum in fermentation of mixed sugar. The method comprises the following steps of: performing gene engineering modification on clostridium acetobutylicum, so that compared with wild type clostridium acetobutylicum, the clostridium acetobutylicum has the advantages that expression of g1cG gene can be inhibited, and the expression and activity of xylose transportprotein, xylose isomerase, and/or xylulokinase can be improved; and applying the obtained clostridium acetobutylicum which is subjected to gene engineering to fermentation of sugar. By the method, more xylose and arabinose can be used by clostridium acetobutylicum in the fermentation of the mixed sugar, a solvent product with high concentration can be produced, and the product yield can be improved; and the method has excellent industrial application prospect.

Description

A kind of method that improves clostridium acetobutylicum sugared utilization ratio in mixing sugar fermentation

Technical field

The invention belongs to genetic engineering technique and fermentation technical field.Particularly, the present invention relates to the method for a kind of raising clostridium acetobutylicum (Clostridium acetobutylicum) sugared utilization ratio (the especially utilization ratio of wood sugar and pectinose) in mixing sugar fermentation, bacterial strain, its purposes and the preparation method for the method.

Background technology

Butanols is the large basic material serving many purposes, and in the chemical fields such as dyestuff, paint, plastics, resin, rubber, can be used as the synthetic precursor of multiple organic compound; For requisite solvent in microbiotic and synthetic drug production process; Also be the food grade extractant of food, perfume industry simultaneously.On the other hand, butanols be still a kind of octane value higher than high-grade fuel and the fuel dope of gasoline, its high boiling point (118 ℃) and low-steam pressure contribute to the cold start-up of automobile; And because the hydrophobicity of butanols is stronger than ethanol, it is easier to vapour, diesel hydrocarbon class A fuel A miscible; In addition, the perfect combustion of butanols, can reduce the CO of tail gas greatly ₂discharge, and there is not residual hydrocarbons pollution, very favourable to purifying air.Obviously, above-mentioned advantage likely makes butanols become the novel green fuel of alternate-engine, and substituting mineralising fuel becomes one of renewable energy resources of Sustainable development, in following transport fuel structure, will occupy important proportion.

It is substrate that the production bacterium clostridium acetobutylicum (Clostridium acetobutylicum) that the traditional butylic fermentation of China adopts in producing be take grain raw material (as corn, wheat etc.).Higher provision price causes expense of raw materials to account for the ratio higher (more than 75%) of solvent total production cost, and this has not only limited the butanols competitiveness of product in market, has also seriously run counter to the grain security strategy of China.Therefore,, with regard to long-range, with non-grain raw material, especially cheap lignocellulose resource (as stalk, straw etc.) manufactures by bio-transformation the inexorable trend that butanols is Future Development.

Clostridium acetobutylicum, except utilizing glucose, sucrose, starch, can also utilize the several kinds of carbon source such as wood sugar, lactose, pectinose.Mierocrystalline cellulose and the main component after hydrolysis of hemicellulose in agriculture and forestry organic waste material (stalk, straw etc.) are glucose, wood sugar and pectinose, and the wide in range substrate of clostridium acetobutylicum spectrum makes this bacterium can utilize Mierocrystalline cellulose, hemicellulose hydrolysate to carry out the fermentation of biological butanol for raw material.Mierocrystalline cellulose and hemicellulose account for the more than 50% of vegitabilia's carbon element at occurring in nature, utilize Mierocrystalline cellulose and hemicellulose hydrolysate to carry out biological butanol fermentation, are expected to greatly reduce raw materials cost.

Yet clostridium acetobutylicum and a lot of the same carbohydrate metabolism thing reptation behaviors (carbon catabolite repression, CCR) that exist of other bacterium,, when glucose exists, utilize wood sugar and pectinose hardly.In addition also there is bottleneck in clostridium acetobutylicum xylose metabolism itself.Given this, improve clostridium acetobutylicum wood sugar and arabinose utilization in mixing sugar and need to overcome two problems, the one, when glucose exists to wood sugar, Arabic glycometabolic checking, the 2nd, the bottleneck that xylose metabolism self exists.

PTS system (phosphoenolpyruvic acid-sugar phosphotransferase system, phosphoenolpyruvate-carbohydrate phosphotransferase system) be the Major Systems that produces solvent clostridium transhipment hexose, when its transhipment carbohydrate enters in born of the same parents, follow the phosphorylation of substrate.Typical PTS system comprises phosphoenolpyruvic acid (PEP), HPr (Histidine-can phosphorylated protein, histidine-phosphorylatable protein) and three catalytic entities, is respectively enzyme I, enzyme II and enzyme III.Glc (glucose-the glucoside of enzyme II component, glucose-glucoside) albumen or Man (seminose-fructose-sorbose, mannose-fructose-sorbose) activity of the adjustable glucose PTS of albumen, in clostridium acetobutylicum, proved Glc family protein the phosphorylation of glucose is played a crucial role, gene glcG is wherein predicted in information biology level.

Yet, investigator finds to knock out PTS system and can cause glucose utilization to postpone even not use in for example, research for various bacteria (Bacillus subtilus, intestinal bacteria, streptomycete), and then production efficiency and the transformation efficiency of reduction glucose, therefore think that this system is not suitable for as transformation target spot (Eiteman, M.A., S.A.Lee etc. (2008). j Biol Eng2:3.; Paulsen, I.T., S.Chauvaux, waits (1998). j Bacteriol180 (3): 498-504.; Perez-Redondo, R., I.Santamarta etc. (2010), microbiology156 (Pt 5): 31527-1537.).

Xylose metabolism via xylose isomerase enzyme catalysis in microorganism mainly comprises: 1) wood sugar transports in the export-oriented born of the same parents of born of the same parents by translocator (xylT); 2) in born of the same parents, wood sugar generates 5-phosphoric acid-xylulose by two steps (xylose isomerase (xylA) and xylulokinase (xylB)) catalyzed reaction; 3) 5-phosphoric acid-xylulose enters phosphopentose pathway (pentose phosphate pathway) and carries out metabolism, among this, comprise again 4 key enzymes: transaldolase, transketolase, 5-phosphoric acid-ribose isomerase and 5-phosphoric acid-ribulose epimerase, last metabolism stream enters glycolytic pathway (referring to Fig. 8).

The common experience of bacterium process as above is catalyzed into glyceraldehyde 3-phosphate by wood sugar and enters center metabolism, and the enzyme of these step reaction of catalysis is respectively Tal, Tkt, Rpe and the Rpi of xylT, xylA, xylB and PPP approach.Although cross express xylose metabolism full gene with improve the strategy of xylose utilization rate in other microorganism, have report (Karhumaa, K., B.Hahn-Hagerdal, et al. (2005). yeast22 (5): 359-368; Zhang, M., C.Eddy etc. (1995). science267 (5195): 240-243.), but in clostridium acetobutylicum, expressing so polygene cannot realize simultaneously at present technically, therefore must confirm real speed limit gene in xylose pathway, reduced the quantity of expressing gene, this work has certain challenge (Nakotte, S., S.Schaffer etc. (1998). appl microbiol Biotechnol50 (5): 564-567; Shao, L., S.Hu etc. (2007). cell Research17 (11): 963-965.).

And according to the pathways metabolism of pectinose (referring to Fig. 8), it is transported to born of the same parents' intracellular metabolite through pectinose translocator is that 5-phosphoric acid-ribulose can be further converted to 5-phosphoric acid-xylulose, and enters thus phosphopentose pathway.Although our research work show xylT (cac1345) be wood sugar translocator (Gu, Y., Y.Ding etc. (2010). bMC Genomics11 (1): 255.), but also there are some researches show it be also pectinose translocator (Servinsky, M.D., J.T.Kiel etc. (2010). microbiology156 (Pt 11): 3478-3491).Therefore, the trans-utilization rate of raising wood sugar may further promote the utilization ratio of pectinose.

In sum, in this area, in the urgent need to developing, a kind ofly improve wood sugar and the method for arabinose utilization and the bacterial strain that can realize the method for clostridium acetobutylicum in mixing sugar fermentation, thereby be conducive to suitability for industrialized production and the application of its tunning (as butanols).

Summary of the invention

The free-revving engine of an aspect of of the present present invention is to provide a kind of method of clostridium acetobutylicum to wood sugar and/or arabinose utilization that improve, thereby can efficiently utilize glucose, wood sugar and/or pectinose fermentative production butanols, acetone and ethanol in raw material.Method of the present invention, can improve by suppressing clostridium acetobutylicum glcG genetic expression, the expression that increases wood sugar translocator, xylose isomerase and xylulokinase or vigor the utilization ratio of wood sugar and/or pectinose.

Another free-revving engine of the present invention is to provide a kind of the have wood sugar of raising and/or the clostridium acetobutylicum of arabinose utilization.Another object of the present invention is to provide the inventive method or the purposes of bacterial strain in butanols, acetone and/or alcohol production.Another object of the present invention is to provide a kind of method of preparing bacterial strain of the present invention.

In a first aspect of the present invention, a kind of method of clostridium acetobutylicum to the utilization ratio of wood sugar and/or pectinose that improve is provided, described method comprises step:

(a) clostridium acetobutylicum is carried out to genetically engineered transformation, with for wild-type clostridium acetobutylicum: suppress the expression of glcG genetic expression, the expression that improves wood sugar translocator or vigor, raising xylose isomerase or expression or the vigor of vigor and/or raising xylulokinase;

(b) fermentation for the raw material containing wood sugar and/or pectinose by the genetically engineered clostridium acetobutylicum of gained in step (a).

In preference of the present invention, to compare with wild-type acetone-butanol shuttle, described genetically engineered acetone-butanol shuttle has at least improved 20% to the utilization ratio of wood sugar, for example 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500% or any interval between them, preferably 50～500%, preferably 55～400%, more preferably 60～300%.

In preference of the present invention, to compare with wild-type acetone-butanol shuttle, the productive rate of described genetically engineered acetone-butanol shuttle fermentative production product acetone, butanols, ethanol has at least improved 10%, for example 20%, 30%, 50%, 80%, 100%, 200%, 300%, 400%, 500% or any interval between them, preferably 10～500%, preferably 20～400%, more preferably 50～400%.

In an embodiment of the invention, described clostridium acetobutylicum is selected from: ATCC 824; EA 2018; Or other can produce the clostridium acetobutylicum of butanols, acetone and ethanol.

In a preference, described clostridium acetobutylicum is ATCC 824.

In yet another embodiment of the present invention, described inhibition glcG genetic expression is to realize by being selected from one or more modes of lower group: in glcG gene, insert DNA fragmentation, partly or entirely knock out glcG gene, introduce antisense nucleic acid or interfere RNA, introducing glcG inhibition; The expression of described raising wood sugar translocator, xylose isomerase and/or xylulokinase or vigor are to realize by being selected from one or more modes of lower group: in acetone-butanol clostridium gene group, import extra wood sugar translocator, xylose isomerase and/or xylulokinase gene; Introduce and improve wood sugar translocator, xylose isomerase and/or the expression of xylulokinase or the sudden change of vigor; Or provide the expression vector of transient expression wood sugar translocator, xylose isomerase and/or xylulokinase.

In a preference, in glcG gene, inserting DNA fragmentation is by two class Intron insertion technology, in any site of glcG gene inside, insert DNA (for example glcG-targetron) or by homologous recombination, in any site of glcG gene, insert DNA sequence dna and realize.

In one embodiment, described inhibition glcG genetic expression is achieved in the following ways: the 1st of glcG gene, between the 1923rd bit base, insert exogenous dna fragment.Preferably, the 1st of glcG gene, between the 1761st bit base, or the 1st between 1554 bit bases, or the 1st between 1248 bit bases, or between the 1st to 270, or insert exogenous dna fragment between 269 to 270.

In another preference, the glcG genetic expression of described genetically engineered clostridium acetobutylicum is suppressed, wood sugar translocator is crossed expression, xylose isomerase is crossed expression and/or xylulokinase is crossed expression.

In another preference, described glcG genetic expression suppressed comprising: do not express that glcG, glcG expression amount decline, being beyond expression has the glcG of complete structure and/or function.

In yet another embodiment of the present invention, described wood sugar translocator be come from can utilize wood sugar organism, for albumen or its bioactive fragment of wood sugar transhipment, or described albumen or its bioactive fragment are through replacement, disappearance or the interpolation of one or more amino-acid residues and form the aminoacid sequence still with transhipment wood sugar function; Described xylose isomerase be come from can utilize wood sugar organism, for enzyme or its bioactive fragment of catalysis wood sugar generation isomery, or described albumen or its bioactive fragment are through replacement, disappearance or the interpolation of one or more amino-acid residues and form the aminoacid sequence still with catalysis wood sugar generation isomery function; Described xylulokinase be come from can utilize wood sugar organism, for enzyme or its bioactive fragment of catalysis xylulose phosphate, or described albumen or its bioactive fragment are through replacement, disappearance or the interpolation of one or more amino-acid residues and form the aminoacid sequence still with catalysis xylulose phosphate function.

In yet another embodiment of the present invention, described organism is selected from: clostridium acetobutylicum, intestinal bacteria, Agrobacterium, pseudomonas, acetobacter, gluconobacter sp, root nodule bacterium, yellow sporangium, Klebsiella pneumoniae, Escherichia, red bacterium, Flavobacterium or Salmonellas.

In yet another embodiment of the present invention, described wood sugar translocator is by xylT genes encoding; Xylose isomerase is by xylA genes encoding; Xylulokinase is by xylB genes encoding.In a preference, described xylT gene is selected from: CA_C1345, CEA_G1359 or with the molecule of described sequence hybridization or with above-mentioned molecule, have the molecule of 90% above homology under stringent condition; Described xylB gene is selected from: CA_C2612, CEA_G2621 or with the molecule of described sequence hybridization or with above-mentioned molecule, have the molecule of 90% above homology under stringent condition; Described xylA gene is selected from: CA_C2610, CEA_G2619 or with the molecule of described sequence hybridization or with above-mentioned molecule, have the molecule of 90% above homology under stringent condition.

In another preference, with the comparison of wild-type clostridium acetobutylicum, the glcG genetic expression of the clostridium acetobutylicum of genetically engineered transformation is suppressed 20 ~ 100%, and preferably 35 ~ 100%, preferably 50 ~ 100%, more preferably 75～100%; Expression or the vigor of HUCEP-8 have improved 50 ~ 200%, and more preferably 75～150%; The expression of xylose isomerase or vigor have at least improved 20%, and for example 30%, 50%, 80%, 100%, 200%, 300%, 400%, 500%, 1000% or any interval between them, preferably 20～1000%, preferably 50～500%, more preferably 75～500%; And/or the expression of xylulokinase or vigor improved and at least improved 20%, for example 30%, 50%, 80%, 100%, 200%, 300%, 400%, 500%, 1000% or any interval between them, preferably 20～1000%, preferably 50～500%, more preferably 75～500%.

In yet another embodiment of the present invention, described genetically engineered clostridium acetobutylicum transforms to be selected from one or more plasmids of lower group: pWJ1-glcG, pIMP1-th1-xylT, pIMP1-th1-xylA, pIMP1-th1-xylB, pIMP1-th1-xylT-th1-xylBA, pIMP1-th1-xylT-th1-xylA, pIMP1-th1-xylT-th1-xylB, pIMP1-th1-xylBA or pIMP1-th1-xylT-th1.In another preference, can adopt any th1 promotor that the insensitive promotor of glucose is substituted in above-mentioned plasmid to build plasmid, preferably ptb, adc promotor.

In a preference, described genetically engineered clostridium acetobutylicum is selected from: clostridium acetobutylicum (pIMP1-thl-xylT), clostridium acetobutylicum (pIMP1-thl-xylA), clostridium acetobutylicum (pIMP1-thl-xylB), clostridium acetobutylicum (pIMP1-thl-xylBA), and clostridium acetobutylicum (pIMP1-thl-xylT-thl-xylBA), clostridium acetobutylicum glcG, clostridium acetobutylicum glcG (pIMP1-thl), clostridium acetobutylicum glcG (pIMP1-thl-xylT), clostridium acetobutylicum glcG (pIMP1-thl-xylA), clostridium acetobutylicum glcG (pIMP1-thl-xylB), with clostridium acetobutylicum glcG (pIMP1-thl-xylT-thl-xylBA).In another preference, can adopt any th1 promotor that the insensitive promotor of glucose is substituted in above-mentioned plasmid to build plasmid, and prepare genetically engineered bacterium with this plasmid, preferably ptb, adc promotor.

In yet another embodiment of the present invention, the raw material that comprises wood sugar and/or pectinose described in is selected from: the hydrolyzed solution of Mierocrystalline cellulose or hemicellulose, grain, cotton etc.

In a preference, described Mierocrystalline cellulose or hemicellulose be available from agriculture and forestry organic waste material, preferably the non-grain raw material such as stalk, straw.

In another preference, the hydrolysis of described Mierocrystalline cellulose or hemicellulose is to be hydrolyzed or the method for biological enzyme hydrolysis is carried out by chemical method.

In another preference, in described raw material, also comprise glucose, preferably comprise glucose-wood sugar-pectinose, glucose-wood sugar.

In another preference, in described raw material, also comprise glucose, preferably comprise glucose-wood sugar-pectinose, glucose-wood sugar, the 5%(that wherein content of wood sugar is not less than total reducing sugar amount in raw material preferably 6%, 8%, 10%)

In a second aspect of the present invention, a kind of genetically engineered clostridium acetobutylicum is provided, it is compared with wild-type clostridium acetobutylicum, has one or more features that are selected from lower group: glcG genetic expression is suppressed or glcG protein vigor is suppressed, wood sugar translocator is crossed expression or vigor improves, xylose isomerase is crossed expression or vigor improves and/or xylulokinase is crossed expression or vigor raising.

In an embodiment of the invention, by being selected from, one or more the genetically engineered processing of lower group obtains described genetically engineered clostridium acetobutylicum: in glcG gene, insert DNA fragmentation, partly or entirely knock out glcG gene, introduce for the antisense nucleic acid of glcG gene or interfere RNA, introducing glcG inhibition; Import extra wood sugar translocator, xylose isomerase and/or xylulokinase gene; Introduce and improve wood sugar translocator, xylose isomerase and/or the expression of xylulokinase or the sudden change of vigor; Or provide the expression vector of transient expression wood sugar translocator, xylose isomerase and/or xylulokinase.

In another preference, in glcG gene, inserting DNA fragmentation is by two class Intron insertion technology, in any site of glcG gene inside, insert DNA or by homologous recombination, in any site of glcG gene, insert DNA sequence dna and realize.

In yet another embodiment of the present invention, described genetically engineered clostridium acetobutylicum is that the clostridium acetobutylicum based on being selected from lower group builds: ATCC 824 or EA2018, should understand those of ordinary skills and can adopt known any clostridium acetobutylicum.In a preference, described clostridium acetobutylicum is ATCC 824.

In another preference, described genetically engineered clostridium acetobutylicum transforms to be selected from one or more plasmids of lower group: pWJ1-glcG, pIMP1-th1-xylT, pIMP1-th1-xylA, pIMP1-th1-xylB, pIMP1-th1-xylT-th1-xylBA, pIMP1-th1-xylT-th1-xylA, pIMP1-th1-xylT-th1-xylB, pIMP1-th1-xylBA or pIMP1-th1-xylT-th1.In another preference, can adopt any th1 promotor that the insensitive promotor of glucose is substituted in above-mentioned plasmid to build plasmid, preferably ptb, adc promotor.

In a third aspect of the present invention, method of the present invention or the purposes of genetically engineered clostridium acetobutylicum of the present invention in the production of butanols, acetone and/or ethanol are provided.

In a preference, described production is fermentative production, for the raw material packet of fermenting, containing one or more of glucose, wood sugar, pectinose, preferably comprises glucose-wood sugar-pectinose, glucose-wood sugar.

In another preference, for the raw material that ferments, available from the hydrolyzed solution of Mierocrystalline cellulose or hemicellulose, grain, preferred described Mierocrystalline cellulose or hemicellulose be available from agriculture and forestry organic waste material, more preferably the non-grain raw material such as stalk, straw.

In a fourth aspect of the present invention, a kind of method of preparing genetically engineered clostridium acetobutylicum used in the inventive method or said gene through engineering approaches clostridium acetobutylicum of the present invention is provided, and described method comprises and clostridium acetobutylicum is selected to one or more genetically engineered transformations of lower group: in glcG gene, insert DNA fragmentation, partly or entirely knock out glcG gene, introduce for the antisense nucleic acid of glcG gene or interfere RNA, introducing glcG inhibition; Introduce and improve wood sugar translocator, xylose isomerase and/or the expression of xylulokinase or the sudden change of vigor; Import extra wood sugar translocator, xylose isomerase and/or xylulokinase gene; Or provide the expression vector of transient expression wood sugar translocator, xylose isomerase and/or xylulokinase.

In another preference, described method also comprises carries out enlarged culturing and/or preservation to the genetically engineered clostridium acetobutylicum of gained.

With the ordinary method comparison that adopts wild-type clostridium acetobutylicum, adopt method of the present invention or adopt genetically engineered clostridium acetobutylicum provided by the present invention, can make clostridium acetobutylicum, in fermentation, wood sugar and/or arabinose utilization be improved to 20～200%, preferably 25～150%.

With the ordinary method comparison that adopts wild-type clostridium acetobutylicum, adopt method of the present invention or adopt genetically engineered clostridium acetobutylicum provided by the present invention, the productive rate that can make clostridium acetobutylicum produce product acetone, butanols, ethanol in the material fermentation that contains wood sugar/pectinose at least improves 10%, for example 20～500%, preferably 10～500%, preferably 20～400%, more preferably 50～400%.

Other side of the present invention, due to disclosure herein, is apparent to those skilled in the art.

Accompanying drawing explanation

Figure 1A is depicted as 4% glucose: the 2% pectinose P2 fermentation clostridium acetobutylicum ATCC824glcG of 0-89hr and the residual sugar content detection result of clostridium acetobutylicum ATCC824; Figure 1B is depicted as 4% glucose: the 2% wood sugar P2 fermentation clostridium acetobutylicum ATCC824glcG of 0-89hr and the residual sugar content detection result of clostridium acetobutylicum ATCC824.

Figure 2 shows that glcG interrupts the detected through gel electrophoresis result of the bacterium colony PCR evaluation of bacterial strain, wherein, NC represents not add the negative control of template, WT template used is clostridium acetobutylicum ATCC824 genome, the template of glcG mutant is respectively transformant 824glcG1-5, and molecular weight marker is 1kb DNA ladder degree.

Figure 3 shows that 4% glucose: clostridium acetobutylicum ATCC824,824glcG, 824glcG-thl, 824glcG-xylT, 824glcG-xylA, 824glcG-xylB and the 824glcG-TBA glucose of 2% wood sugar P2 fermentation 96hr, the detected result of wood sugar consumption.

Figure 4 shows that the detected result that imports pIMP 1-thl-xylT-thl-xylBA in clostridium plasmid PCR evaluation 824glcG, wherein, A, B represent two pairs of primers designed of different positions, "-" represents to take the negative control that water is template amplification, "+" represents take the positive control that the plasmid that builds is template amplification, 1～6 represents the plasmid of different strains extracting, and No. 6 positive.

Figure 5 shows that 3.8% glucose: 1.4% wood sugar: the residual sugar content detection result of 0.3% pectinose fermentation 824 and 824glcG-TBA.

Figure 6 shows that 3.8% glucose: 1.4% wood sugar: 0.3% pectinose fermentation 824 and the butanols of 824glcG-TBA and the detected result of ABE.

Figure 7 shows that the residual sugar content detection result of 1.5% pectinose and 1.5% wood-sugar fermentation 824.

Figure 8 shows that the schematic diagram of bacterium pectinose and xylose metabolism.

Figure 9 shows that 4% glucose: the residual sugar content detection result of 2% wood-sugar fermentation 824 and 824-xylBA.

Figure 10 shows that 4% glucose: the residual sugar content detection result of 2% wood-sugar fermentation 824 and 824-xylT.

Figure 11 shows that 4% glucose: the glucose of 2% wood-sugar fermentation 2018,2018glcG and 2018glcG-TBA 96hr, the detected result of wood sugar consumption.

Figure 12 shows that 4% glucose: the solvent detected result of 2% wood-sugar fermentation 2018,2018glcG and 2018glcG-TBA 96hr.

Embodiment

The inventor is through long-term and deep research, developed and a kind ofly improved wood sugar in clostridium acetobutylicum fermenting process and/or the method for arabinose utilization, thereby can efficiently utilize glucose, wood sugar and/or pectinose in raw material (as Mierocrystalline cellulose or hemicellulose hydrolysate) independent or fermentative production butanols, acetone and ethanol altogether.Method of the present invention mainly realizes by suppressing the expression of clostridium acetobutylicum glcG genetic expression and increase wood sugar translocator, xylose isomerase and xylulokinase.For example, in a preferred embodiment of the present invention, by inserting DNA fragmentation and import pIMP1-thl-xylT-thl-xylBA in clostridium acetobutylicum glcG gene, can realize the present invention.

The inventor also further provides the clostridium acetobutylicum recombinant bacterial strain for aforesaid method, wherein suppressed, the xylT gene overexpression of clostridium acetobutylicum glcG genetic expression, xylA gene overexpression and/or xylB gene overexpression.For example, in a preferred embodiment of the present invention, thereby the glcG gene in the genome of recombinant bacterial strain provided by the invention has inserted DNA fragmentation, cause its expression suppressed, and this bacterial strain is also crossed xylT gene, xylA gene and the xylB gene of expressing.

Particularly, in prior art, in many microorganisms (as withered grass, large intestine, streptomycete), find that knocking out PTS system all can cause glucose utilization to postpone not even use, therefore think that this target spot is not desirable transformation target spot.Yet, although contriver decreases earlier fermentation glucose utilization speed by knocking out glcG in this research, but the fermentation period of glucose does not postpone (for example, referring to Figure 1A and 1B), not to sacrifice the utilization that is used to exchange for the secondary carbon sources such as wood sugar, pectinose of glucose.And contriver further studies discovery, the reason that glucose utilization does not have to postpone is that the activation of the non-PTS system of glucose has compensated glucose transport and follow-up phosphorylation.This unique phenomenon that contriver finds in clostridium acetobutylicum makes glcG gene become the important transformation target spot of eliminating this microorganism glucose retarding effect.

And bacterium changes into glyceraldehyde 3-phosphate by wood sugar and enters center metabolism and need pass through the catalysis of a plurality of steps, plurality of enzymes.Although cross, express xylose pathway full gene with the strategy that improves xylose utilization rate report to some extent in other microorganism, but in clostridium acetobutylicum, expressing so polygene cannot realize simultaneously at present technically, therefore must confirm real speed limit gene in xylose pathway, reduced the quantity of expressing gene, this work has certain challenge.The inventor finds that by research xylose metabolism bottleneck is the three-step reaction of xylT, xylA and xylB catalysis.Cross respectively and express and cross and express this three genes simultaneously, really improved the xylose utilization rate of thalline in mixing sugar fermentation.The research result makes the strategy of strengthening xylose metabolism approach in clostridium acetobutylicum become possibility, and the structure of engineering bacteria is had to great importance.

Method of the present invention and bacterial strain can be used for efficiently utilizing the application of glucose-wood sugar-pectinose mixing sugar fermentative production butanols, acetone and ethanol, have wide industrial application and market outlook.

As used herein, term " clostridium acetobutylicum " refers to that a kind of gram's staining is positive, cell is shuttle shape and can produce the genus bacillus of the materials such as acetone and butanols.This bacterioid energy decomposing protein and carbohydrate, and produce a large amount of acetone, butanols and ethanol equal solvent in starchiness and saccharine material fermentation, be important industrial fermentation bacterial classification.In this area, there are many documents to describe to some extent the kind of this bacterioid and function, Keis for example, S. etc., Int.J.Syst.Bacteriol., 45,693,1995; Keis, S. etc., Int.J.Syst.Evol.Microbiol., 51,2095,2001. etc.Although the application's embodiment part be take ATCC 824 and EA2018 as example has been described in detail the present invention, should understand can adopt in the present invention and anyly meet clostridium acetobutylicum defined above, and be not limited to embodiment.

As used herein, term " productive rate " refers to that the output of resultant is than the per-cent that drops into material quantity.In this article, refer to that the growing amount of ethanol, butanols and/or acetone is than the per-cent that drops into the amount of total reducing sugar in raw material, wherein total reducing sugar refers to all sugar comprising in raw material, includes but not limited to glucose, wood sugar and/or pectinose in raw material.

As used herein, term " utilization ratio " refers to that the amount of the raw material that clostridium acetobutylicum consumes is than the per-cent that drops into the amount of raw material.

As used herein, term " raising productive rate ", or " increasing operation rate " be relative wild type strain, specifically refers to than wild type strain and have higher productive rate or higher utilization ratio.

the expression inhibiting of glcG gene or the inhibition of protein-active

In the present invention, indication " suppressing clostridium acetobutylicum glcG genetic expression ", can be both to reduce clostridium acetobutylicum glcG gene expression amount, can be also clostridium acetobutylicum glcG gene not to be expressed maybe can not give expression to correct protein.The sequence of known glcG gene in this area (CA_C (0570)), its coding PTS enzyme II, containing A, B, tri-catalytic subunits of C, thereby arbitrary disappearance or the suppressed inhibition to the whole expression of glcG and vigor causing in these three catalytic subunits in the present invention, have also been comprised.

The method that realizes above-mentioned purpose in the art by those skilled in the art known (such as but not limited to Shao, L., S.Hu etc. (2007). cell Research17 (11): 963-965; The people < < molecular clonings such as Sambrook: lab guide > > (New York:Cold Spring Harbor Laboratory Press, 1989)), at this, do not specifically describe.For example, according to a preferred embodiment of the present invention, by interrupting clostridium acetobutylicum glcG genetic expression, can improve the utilization ratio of clostridium acetobutylicum to wood sugar and/or pectinose.In another preferred embodiment of the present invention, in the genome of the clostridium acetobutylicum recombinant bacterial strain providing glcG genetic expression suppressed, can not give expression to the glcG albumen with complete structure.

According to the present invention, the interruption of clostridium acetobutylicum glcG gene, can adopt two class Intron insertion technology in any site of this gene inside, to insert DNA (for example intron or size are no more than the resistant gene of 1kb, as the erythromycin resistance gene on pIMP1 carrier framework) realizes; Also can interrupt glcG gene by homologous recombination, for interrupting the Insert Fragment of glcG gene, can insert in any site of glcG gene, only need to make glcG genetic expression be interrupted or suppress; Can also knock out the part or all of sequence of glcG by homologous recombination and realize, as long as can make the expression of glcG gene be interrupted or suppressed or give expression to imperfect glcG albumen.Aforesaid method can be used for the gene inactivation by glcG.

In addition, glcG protein structure predictive display for clostridium acetobutylicum, this protein comprises A, B, tri-catalytic subunits of C, wherein the 1st is first catalytic subunit C to the 416th amino acids (corresponding the 1st to the 1248th bit base), the 417th is the second catalytic subunit B to 518 amino acids (corresponding the 1249th to the 1554th bit base), and the 519th is the 3rd catalytic subunit C to the 641st amino acids (corresponding the 1555th to the 1923rd bit base).The halfcystine of the 437th that is positioned at glcG albumen the second catalytic subunit is crucial conservative site (Martin Tangney, Wilfrid J.Mitchell. (2007) .Appl Microbiol Biotechnol (74): 398-405.) with the Histidine of the 587th that is positioned at glcG albumen the 3rd catalytic subunit.Therefore, in the preferred embodiment of the present invention, at the 1-1923 bit base insertion exogenous dna fragment of glcG gene.In another preferred implementation of the present invention, at the 1st, between the 1761st bit base, insert exogenous dna fragment, or insert exogenous dna fragment between the 1st and the 1554th, or insert exogenous dna fragment between the 1st and the 1248th, or insert exogenous dna fragment between the 1st and the 270th.In a specific embodiment of the present invention, between the 269/270th of glcG gene, insert DNA fragmentation.

In addition, can also adopt this area routines such as antisense technology for suppressing the method for specific gene expression, to suppress the expression of glcG gene, glcG expression amount is lowered.The method that realizes above-mentioned purpose in the art by those skilled in the art known (such as but not limited to Tummala, S.B., N.E.Welker etc. (2003). j Bacteriol185 (6): 1923-1934; The people < < molecular clonings such as Sambrook: lab guide > > (New York:Cold Spring Harbor Laboratory Press, 1989)), therefore at this, do not specifically describe.

" restructuring knocks out plasmid vector " refers to for knocking out the recombinant plasmid vector of glcG gene, this carrier is interpreted as having the recombinant plasmid vector that carries out special pairing site with the particular sequence of glcG gene, at above-mentioned recombinant plasmid vector, comprises for glcG gene is carried out to the fragment that specificity knocks out.

In the preferred embodiments of the present invention, the restructuring of using knocks out plasmid vector pWJ1-glcG and refers to: based on intestinal bacteria and clostridium acetobutylicum shuttle plasmid pWJ1, (it expresses erythromycin resistance gene in clostridium acetobutylicum, sequence is as shown in SEQ ID NO.:1) build, for knocking out the recombinant plasmid vector of glcG gene.In this carrier, the glcG-targetron fragment of using refers at IBS, EBS2, EBS 1d site base is after revising, for knocking out the fragment of glcG gene, this fragment belongs to a L1.LtrB intron part, and described L1.LtrB bis-class introns are protokaryon two class introns, wherein comprise ltrA gene.Yet, those of ordinary skills are when putting into practice method of the present invention, can choose other insertion point and test, even can also not use recombinant plasmid vector to test, as long as can insert nucleic acid fragment to interrupt the expression of glcG gene in glcG gene.

Should be understood that except can suppressing the expression of glcG gene from gene level, in order to realize object of the present invention, also can suppress the activity of glcG albumen.

the rise of xylT, xylA, xylB genetic expression or protein vigor

According to the present invention, " raising genetic expression ", " genetic expression rise " or " cross and express " can be both the expression amounts that improves goal gene (being xylT, xylA and/or xylB gene), can be also to make destination gene expression go out the target protein that vigor improves.

The method that realizes above-mentioned purpose in the art by those skilled in the art known (such as but not limited to Sillers, R., M.A.Al-Hinai, et al. (2009). biotechnology and Bioengineering102 (1): 38-49; The people < < molecular clonings such as Sambrook: lab guide > > (New York:Cold Spring Harbor Laboratory Press, 1989)), at this, do not specifically describe.

For example, can in acetone-butanol clostridium gene group, import extra (as one or more copy) wood sugar translocator, xylose isomerase and/or xylulokinase gene; Introduce and improve wood sugar translocator, xylose isomerase and/or the expression of xylulokinase or the sudden change of vigor; Or provide the expression vector of transient expression wood sugar translocator, xylose isomerase and/or xylulokinase.

As used herein, term " wood sugar translocator " refers to and comes from albumen or its bioactive fragment that can be used for wood sugar transhipment in any organism (especially mushroom) that can utilize wood sugar, and described organism can be such as (but being not limited to): clostridium acetobutylicum, intestinal bacteria, Agrobacterium, pseudomonas, acetobacter, gluconobacter sp, root nodule bacterium, yellow sporangium, Klebsiella pneumoniae, Escherichia, red bacterium, Flavobacterium, Salmonellas etc.Can be referring to for example about the description of wood sugar translocator and encoding sequence thereof: Jojima, T. etc. (2010) .Appl Microbiol Biotechnol 85 (3): 471-480; Sumiya, the 1995.ReceptorsChannels 3:117-28 such as M.; Henderson, P.J.1990.J Bioenerg Biomembr 22:525-69; Leandro, the 2006.Biochem J 395:543-9 such as M.J.; Saloheimo, the 2007.Appl Microbiol Biotechnol 74:1041-52 such as A.; Weierstall, the 1999.Mol Microbiol 31:871-83 such as T.; Tarr, the 2009.Biochim Biophys Acta 1791:584-93 such as P.T.; Davis, the 1987.J Biol Chem262:13928-32 such as E.O.; Schneider, E.2001.Res Microbiol 152:303-10.

As used herein, term " xylose isomerase " refers to and comes from any organism (especially mushroom) that can utilize wood sugar for enzyme or its bioactive fragment of catalysis wood sugar generation isomery, and described organism can be such as (but being not limited to): clostridium acetobutylicum, intestinal bacteria, Agrobacterium, pseudomonas, acetobacter, gluconobacter sp, root nodule bacterium, yellow sporangium, Klebsiella pneumoniae, Escherichia, red bacterium, Flavobacterium, Salmonellas etc.Can be referring to for example about the description of xylose isomerase and encoding sequence thereof: CN102037120A (table 1 especially wherein).

As used herein, term " xylulokinase " refers to and comes from any organism (especially mushroom) that can utilize wood sugar for enzyme or its bioactive fragment of catalysis xylulose phosphate, and described organism can be such as (but being not limited to): clostridium acetobutylicum, intestinal bacteria, Agrobacterium, pseudomonas, acetobacter, gluconobacter sp, root nodule bacterium, yellow sporangium, Klebsiella pneumoniae, Escherichia, red bacterium, Flavobacterium, Salmonellas etc.Can be referring to for example about the description of xylose isomerase and encoding sequence thereof: CN102037120A (table 2 especially wherein).

In the definition of " wood sugar translocator " of the present invention, " xylose isomerase " and " xylulokinase ", also comprise replacement, disappearance or the interpolation through one or more amino-acid residues and form aminoacid sequence and the bioactive fragment thereof still with its original function, for example that the aminoacid sequence in wild-type wood sugar translocator, xylose isomerase or xylulokinase is individual through 1-20, preferably 1-10, more preferably the replacement of 1-5 amino-acid residue, disappearance or interpolation form, and have former activated derived protein.Derived protein can comprise the alternative sequence of a part of conserved amino acid, and the described sequence through amino acid substitution does not affect its activity or retained the activity of its part.Suitably replacing amino acid is technology well known in the art, and described technology can be implemented at an easy rate, and guarantees not change the biological activity of gained molecule.These technology are recognized those skilled in the art, in general, at the inessential area change single amino acids of a peptide species, substantially can not change biological activity.See Watson etc., " Molecular Biology of The Gene " (< < gene molecule biology > >, the 4th edition, 1987, Benjamin/Ku Ming publishing company, The Benjamin/Cummings Pub.Co.P224).

Should understand through the conservative sequence mentioned above replacing and also can be used for the present invention, preferred reactive derivative refers to compare with original acid sequence, has 5 at the most, preferably at the most 3, more preferably at the most 2,1 amino acid is replaced by the similar or close amino acid of character and forms polypeptide best.These conservative property variation polypeptide are preferably replaced and are produced according to conserved amino acid known in the art.

Term " encoding sequence " refers to that code book invents the sequence of above-mentioned albumen or polypeptide, it can be sequence as known in the art (described in the same document), under stringent condition with the molecule of known array hybridization or with the family gene molecule of above-mentioned numberator height homology.

As used herein, term " stringent condition " refers to: (1) at the hybridization compared with under low ionic strength and comparatively high temps and wash-out, as 0.2 * SSC, and 0.1%SDS, 60 ℃; Or (2) hybridization time is added with denaturing agent, as 50% (v/v) methane amide, 0.1% calf serum/0.1%Ficoll, 42 ℃ etc.; Or (3) only the homogeny between two sequences at least 50%, preferably more than 55%, more than 60%, more than 65%, more than 70%, more than 75%, more than 80%, more than 85% or more than 90%, be more preferably 95% and just hybridize when above.For example, described sequence can be the complementary sequence of sequence defined in (a).

Encoding sequence of the present invention or its fragment can obtain by the method for pcr amplification method, recombination method or synthetic conventionally.For pcr amplification method, can be disclosed according to the present invention about nucleotide sequence, especially open reading frame sequence designs primer, and with commercially available cDNA storehouse or by the prepared cDNA storehouse of ordinary method well known by persons skilled in the art as template, amplification and must relevant sequence.When sequence is longer, usually need to carry out twice or pcr amplification repeatedly, and then the fragment that each time amplified is stitched together by proper order.

Should understand, encoding sequence of the present invention is preferably available from clostridium acetobutylicum, available from other bacterium or biology, (as have more than 50% with the encoding sequence height homology available from clostridium acetobutylicum, preferably more than 55%, more than 60%, more than 65%, more than 70%, more than 75%, more than 80%, more preferably more than 85% as 85%, 90%, 95%, even 98% sequence homogeny) the equivalency range also preferably considered in the present invention of other encoding sequence within.The Method and kit for of aligned sequences homogeny is also that this area is known, as BLAST.

According to the present invention, " recombinant plasmid vector " refer to for cross express xylT gene, xylA gene and/or xylB gene plasmid vector.In a preference of the present invention, this carrier is to contain to derive from the promotor of clostridium acetobutylicum ATCC824thl gene and the recombinant plasmid vector of xylT gene, xylA gene and/or xylB gene.In another preference of the present invention, can adopt any the insensitive promotor of glucose (as th1, ptb, adc promotor) to be built to recombinant plasmid vector of the present invention.

For example, in specific examples of the present invention, the recombinant plasmid vector " pIMP1-thl-xylT-thl-xylB " using refers to that (it expresses erythromycin resistance gene in clostridium acetobutylicum based on intestinal bacteria and clostridium acetobutylicum shuttle plasmid pIMP1-thl, sequence is as shown in SEQ ID NO.:2) build, be used for expressing the recombinant plasmid vector of xylT gene and xylB gene, wherein, the promotor of use is the thl gene that derives from clostridium acetobutylicum ATCC824.

In the present invention, can adopt other carrier of similar definition, such as but not limited to: pIMP1-th1-xylT, pIMP1-th1-xylA, pIMP1-th1-xylB, pIMP1-th1-xylT-th1-xylBA, pIMP1-th1-xylT-th1-xylA, pIMP1-th1-xylT-th1-xylB, pIMP1-th1-xylBA etc.

suppress expression or the protein-active of glcG gene and raise xylT, xylA, xylB genetic expression simultaneously or protein vigor

In the method for the invention, more favourable is when suppressing the expression or protein-active of glcG gene, raises the expression of one or more genes or the vigor of one or more albumen in xylT, xylA, xylB.

Can adopt method conventional in this area, realize above-mentioned preferred version.For example, can simultaneously or successively adopt multiple plasmid to transform clostridium acetobutylicum, for example, first adopt pWJ1-glcG to transform, adopt again one or more in pIMP1-th1-xylT, pIMP1-th1-xylA, pIMP1-th1-xylB, pIMP1-th1-xylT-th1-xylBA, pIMP1-th1-xylT-th1-xylA, pIMP1-th1-xylT-th1-xylB, pIMP1-th1-xylBA to transform.

For example, in the present invention, clostridium acetobutylicum glcG (pIMP1-th1-xylT) refers to that with restructuring, knocking out plasmid vector pWJ1-glcG has knocked out glcG gene to suppress this genetic expression, and with recombinant plasmid vector pIMP1-thl-xylT, transforms to realize the recombinant acetone-butanol clostridium bacterial strain of expressing and building of crossing of xylT gene.

Other recombinant bacterial strain that can adopt in the present invention similar method to build, such as but not limited to: clostridium acetobutylicum glcG (pIMP1-thl-xylA), clostridium acetobutylicum glcG (pIMP1-thl-xylB), clostridium acetobutylicum glcG (pIMP1-thl-xylBA), clostridium acetobutylicum glcG (pIMP1-thl-xylT-thl-xylBA) etc.

According to a preferred embodiment of the present invention, in 824glcG, independent mistake is expressed both or the three in xylT, xylA, xylB gene and/or coexpression xylT, xylA, xylB gene, can further improve wood sugar and/or arabinose utilization in the fermentation of clostridium acetobutylicum mixing sugar.

According to the present invention, the recombinant bacterial strain using, both can be inhibition clostridium acetobutylicum glcG provided by the invention genetic expression and the bacterial strain of excessively expressing xylT gene, xylA gene and xylB gene, also can be according to instruction of the present invention and prior art, the inhibition clostridium acetobutylicum glcG genetic expression of other of preparation and cross the bacterial strain express xylT gene, xylA gene and xylB gene, as adopted antisense technology to reduce the bacterial strain of glcG expression amount.

adopt the fermentative production of recombinant bacterial strain of the present invention

Herein, term " ABE " is product acetone, the butanols of clostridium acetobutylicum fermentative production, the abbreviation of ethanol (Acetone-butanol-ethanol), and term " ABE concentration " refers to the total concn of gained acetone, butanols, ethanol.

Method provided by the invention and bacterial strain significantly improve the utilization ratio of the wood sugar in fermentation raw material and/or pectinose, transform the corresponding raising of ABE concentration generating simultaneously, therefore can be used for the fermentative production of acetone, butanols, ethanol.And engineering strain that the present invention builds has improved the rate of consumption of wood sugar and pectinose in mixing sugar, can efficiently utilize glucose-wood sugar-pectinose to ferment, so these bacterial strains have and utilize ligno-cellulose hydrolysate to carry out the potentiality of acetone butanol fermentation.

Herein, term " fermentation " refers to and adopts recombinant acetone-butanol clostridium of the present invention, produced the process of the products such as acetone, butanols, ethanol by sugary raw material by bio-transformation.This process can adopt conventional fermentation equipment and the technique of using in this area to carry out, and those of ordinary skills can select equipment and process with condition according to actual needs.

Raw material for fermentative production of the present invention can be single sugar or mixing sugar, for example wood sugar-pectinose, glucose-wood sugar-pectinose.The sugary raw material using can be both single sugar or the mixing sugar (as glucose-wood sugar-pectinose) of directly using glucose, wood sugar and/or pectinose preparation to obtain, also can be fermentation or hydrolysis macromolecular compound (as hydrocellulose or hemicellulose etc.), the mixing sugar of acquisition.Sugary raw material can be available from traditional grain, but more preferably available from non-grain raw material, cheap lignocellulose resource or agriculture and forestry organic waste material for example, as stalk, straw etc., according to the ratio that is reported in glucose, wood sugar and pectinose in most stalks or straw in prior art document, can be 20～60 quality % glucose: 3～24 quality % wood sugars: 0.5～5 quality % pectinose (Aristidou, A.and M.Penttila (2000) .Curr Opin Biotechnol 11 (2): 187-198; Jeewon Lee, Journal of Biotechnology 56 (1997) 1 – 24).In some embodiments of the present invention, in mixing sugar, the ratio of various sugar can be 2 ~ 5% glucose: 0.3～2% wood sugar: 0.05%～5% pectinose, more preferably 3.9% glucose: 1.5% wood sugar: 0.3% pectinose (shown in per-cent be w/v).

Should understand, although embodiment of the present invention part is only based on the conventional typical module clostridium acetobutylicum ATCC 824 in this area, those of ordinary skills, after having read the present invention, can obtain the sugared utilization ratio of required raising on the basis that does not depart from spirit and scope of the invention to other clostridium acetobutylicum transformation.

advantage of the present invention

Major advantage of the present invention is:

1. in the present invention, found in other bacterium, to be considered to be unfavorable for that the target spot PTS system of transformation can be used for the transformation of clostridium acetobutylicum breakthroughly, made glcG gene become the important transformation target spot of eliminating this microorganism glucose retarding effect;

2. clear and definite speed limit gene real in xylose pathway, by the transformation to xylT, xylA and xylB gene, has improved the xylose utilization rate of thalline in mixing sugar fermentation, and the structure of engineering bacteria is had to great importance;

3. a kind of clostridium acetobutylicum of genetically engineered transformation of novelty is provided, and it can efficiently utilize the essential industry raw material such as wood sugar and/or pectinose production butanols, ethanol and acetone in fermentation raw material;

4. a kind of method that adopts clostridium acetobutylicum of the present invention, efficiently utilizes the essential industry raw materials such as wood sugar and/or pectinose production butanols, ethanol and acetone in fermentation raw material is provided, the method has expanded the optional scope of fermentation raw material, reduce production cost, and there is higher productive rate and efficiency.

Embodiment

Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment are only not used in and limit the scope of the invention for the present invention is described.The experimental technique of unreceipted actual conditions in the following example, conventionally according to normal condition as people < < molecular clonings such as Sambrook: lab guide > > (New York:Cold Spring Harbor Laboratory Press, 1989) condition described in, or the condition of advising according to manufacturer.Unless otherwise indicated, otherwise per-cent and umber calculate by weight.In fermented liquid or sugar soln, the per-cent of sugar is w/v%.

term explanation

Unless otherwise defined, the same meaning that all specialties of using in literary composition and scientific words and one skilled in the art are familiar.In addition, any method similar or impartial to described content and material all can be applicable in the present invention.The use that better implementation method described in literary composition and material only present a demonstration.

In following embodiment of the present invention, glcG, xylT, xylB and xylA gene that the bacterial strain clostridium acetobutylicum Clostridium acetobutylicum ATCC 824 using contains are that prior art is known, and in NCBI nucleic acid database, the sequence number in genome is respectively for it: CA_C0570, CA_C1345, CA_C2612 and CA_C2610.GlcG, xylT, xylB and xylA gene that the bacterial strain clostridium acetobutylicum EA2018 using contains are that prior art is known, and in NCBI nucleic acid database, the sequence number in genome is respectively for it: CEA_G0584, CEA_G1359; CEA_G2621 and CEA_G2619, these sequences are identical with the corresponding sequence of ATCC824.

In following embodiment of the present invention, " ABE " is the abbreviation of acetone, butanols, ethanol (Acetone-butanol-ethanol), and ABE concentration refers to the total concn of acetone in solution, butanols, ethanol.

In following embodiment of the present invention, " 824glcG " refers to, glcG genetic expression suppressed bacterial strain of not even expressing that build based on ATCC 824.

" recombinant plasmid vector pIMP1-thl " refers to the recombinant plasmid vector (its sequence is as shown in SEQ ID NO:2) of expressing xylT, xylB and xylA gene, and thl promotor is the promotor that derives from clostridium acetobutylicum ATCC 824thl gene.

" restructuring knocks out plasmid vector pWJ1-glcG " refers to the recombinant plasmid vector (its sequence is as shown in SEQ ID NO:10) for knocking out glcG gene, wherein, the glcG-targetron fragment of using refers at IBS, EBS2, EBS1d site base is after revising, and for knocking out the fragment of glcG gene, this fragment belongs to a L1.LtrB intron part, described L1.LtrB bis-class introns are protokaryon two class introns, wherein comprise ltrA gene.

bacterial strain and plasmid

Bacterial strain and plasmid that the present invention uses are respectively:

Plasmid pWJ1 is the shuttle plasmid (the replicon pCB102 that derives from clostridium butylicum DSM10702 being replaced to the replicon pIM13 of pSY6) of intestinal bacteria (E.coli) and clostridium acetobutylicum, in clostridium acetobutylicum, express erythromycin resistance gene, the sequence of this plasmid is shown in SEQ ID NO.:1.

Plasmid pIMP1-thl be intestinal bacteria and clostridium acetobutylicum shuttle plasmid (carrier framework is based on reference Mermelstein, L.D.and E.T.Papoutsakis (1993). appl Environ Microbiol59 (4): the pIMP1 in 1077-1081., difference has only been to introduce the promotor of thl gene (cac2873)), in clostridium acetobutylicum, express erythromycin resistance gene, the sequence of this plasmid is shown in SEQ ID NO.:2.

Plasmid pANS1, sequence see SEQ ID NO.:3 (Mermelstein, L.D and E.T.Papoutsakis (1993). appl Environ Microbiol59 (4): 1077-1081.), containing spectinomycin resistance gene.

Bacterial strain intestinal bacteria ER2275 is purchased from New England Biolabs company.

Bacterial strain clostridium acetobutylicum ATCC 824 is purchased from ATCC company.Bacterial strain clostridium acetobutylicum EA 2018 comes from patent ZL 95111733.5, the icp gene group group of it and ATCC824 is studied referring to document Hu, S.Y., H.J.Zheng (2011). " Comparative genomic and transcriptomic analysis revealed genetic characteristics related to solvent formation and xylose utilization in Clostridium acetobutylicum EA 2018. " BMC Genomics 12.

reagent

The PCR purifying using in the present invention and DNA gel reclaim purification kit all purchased from Hua Shun biological products company limited, Targetron ^tMgene Knockout System (TA0100) Kit is purchased from Sigma-Aldrich company, and genome extraction agent box is purchased from Shanghai Sheng Gong biotechnology company limited.

In following embodiment of the present invention, substratum and the damping fluid of use are as follows:

CGM culture medium prescription following (P681-694, Vol 557,1985 for Joseph W.Roos etc., Biotechnology and Bioengineering): 2g (NH ₄) ₂sO ₄, 1g K ₂hPO ₄3H ₂o, 0.5g KH ₂pO ₄, 0.1gMgSO ₄7H ₂o, 0.015g FeSO ₄7H ₂o, 0.01g CaCl ₂, 0.01g MnSO ₄h ₂o, 0.002gCoCl ₂, 0.002g ZnSO ₄, 2g Tryptones, 1g yeast extract (Yeast Extraction), 50g glucose, 2% agar is dissolved in 1L water.

The compound method following (Issue 6, Vol44,1982 for F.Monot etc., Appl Environ Microbiol) of P2 substratum:

Solution 1:40g D-Glucose: 20g D-wood sugar or 40g D-Glucose: 20g L-arabinose, adds H ₂o is dissolved to 850mL;

Solution 2:NH ₄ac 2.2g, K ₂hPO ₄3H ₂o 0.5g, KH ₂pO ₄0.5g, adds H ₂o is dissolved to 100mL;

Solution 3:2.0g MgSO ₄7H ₂o, 0.1g MnSO ₄h ₂o, 0.1g NaCl, 0.1g FeSO ₄7H ₂o;

In solution 4:100ml distilled water, add 100mg benzaminic acid (p-aminobenzoic acid), 100mg VITMAIN B1 (thiamine), 1mg vitamin H (biotin);

Solution 1 and solution 2 high-temperature heat sterilizations, solution 3 and solution 4 filtration sterilizations, mix after solution 1 and solution 2 are cooling, then add 10mL solution 3 and 1mL solution 4, is distributed into 95mL/ bottle after mixing, with filtration sterilization, N ₂get rid of the air in bottle.

The P2 substratum compound method of imitating hydrolyzed solution sugar ratio 5.5%w/v in fermentor tank is as follows:

Solution 1:56.4g D-Glucose, 21.7g D-wood sugar and 4.3g L-arabinose, add H ₂o is dissolved to 1240mL;

Solution 2:NH ₄ac 3.3g, K ₂hPO ₄3H ₂o 0.75g, KH ₂pO ₄0.75g, adds H ₂o is dissolved to 150mL;

Solution 3:2.0g MgSO ₄7H ₂o, 0.1gMnSO ₄h ₂o, 0.1gNaCl, 0.1g FeSO ₄7H ₂o;

Solution 4: add 100mg benzaminic acid (p-aminobenzoic acid) in 100ml distilled water, 100mg VITMAIN B1 (thiamine), 1mg vitamin H (biotin);

Solution 1 and solution 2 high-temperature heat sterilizations, solution 3 and solution 4 filtration sterilizations, solution 1 after sterilizing, leads to N together with fermentor tank ₂cooling, then mix with solution 2, then add 15mL solution 3 and 1.5mL solution 4.

ETM buffer formulation is as follows: 270mM sucrose, 0.6mM Na ₂hPO ₄, 4.4mM NaH ₂pO ₄, 10mM MgCl ₂.

ET buffer formulation is as follows: 270mM sucrose, 0.6mM Na ₂hPO ₄, 4.4mM NaH ₂pO ₄.

The restriction enzyme that the present invention uses, Taq archaeal dna polymerase, T4DNA ligase enzyme and calf alkaline phosphatase (CIAP) are all purchased from TaKaRa company, and KOD plus archaeal dna polymerase is purchased from Toyobo company.

Other conventional reagent is domestic or import packing.

sequence table explanation

In sequence table, SEQ ID NOs:1-38 represents respectively following sequence:

The implication of sequence in table 1. sequence table

* up=upstream primer * * dn=downstream primer

embodiment general introduction

The present invention is used for expressing xylT by pcr amplification, the targetron segment of the fragment of xylB and xylA gene and interruption glcG gene, then through double digestion, and be connected with pIMP1-thl or the pWJ1 carrier cut through same enzyme, obtain plasmid pIMP1-thl-xylT-thl-xylBA and pWJ1-glcG, electricity turns clostridium acetobutylicum ATCC824glcG and clostridium acetobutylicum ATCC 824 or EA 2018, then through clostridium plasmid PCR, identify the existence of exogenous genetic fragment and Intron insertion to the recombinant bacterium in genome, through fermentation checking, determine recombinant bacterium wood sugar in mixing sugar, pectinose rate of consumption improves, specific as follows stating shown in embodiment.

embodiment 1. builds pWJ1-glcG plasmid vector

By pcr amplification glcG targetron segment, then use XhoI and BsrG I to carry out double digestion, and be connected with the pWJ1 carrier of cutting with BsrG I enzyme through XhoI equally, obtain interrupting plasmid pWJ1-glcG, wherein, the template of pcr amplification glcG targetron and primer design method derive from the Targetron of Sigma-Aldrich company ^tMgene Knockout System (TA0100) test kit, concrete steps are as follows:

1.1PCR amplimer

With reference to Targetron ^tMthe method that Gene Knockout System (TA0100) test kit provides, design respectively primer glcG-IBS (as shown in sequence SEQ ID NO.:4), glcG-EBS1d (as shown in sequence SEQ ID NO.:5) and glcG-EBS2 (as shown in sequence SEQ ID NO.:6), for building pWJ1-glcG plasmid vector.

The EBS universal primer (EBS universal) that pcr amplification needs is by Targetron ^tMgene Knockout System (TA0100) test kit carries.

1.2PCR amplification

Use the Targetron of Sigma-Aldrich ^tMgene Knockout System (TA0100) test kit carries out pcr amplification (PCR reaction conditions: 94 ℃ of 30s, 94 ℃ of 30s, 55 ℃ of 30s, 30 circulations of 72 ℃ of 30s, 72 ℃ of 2min, 4 ℃ of preservations), template and reagent that amplification needs are provided by test kit, PCR product is carried out to agarose gel electrophoresis, then use the glue of Hua Shun company to reclaim the band that test kit purifying reclaims 350bp place.

1.3 build pWJ1-glcG recombinant plasmid vector

Use Xho I and BsrGI respectively enzyme cut carrier pWJ1 and glcG-targetron fragment, then use the glue recovery test kit purifying recovery enzyme of Hua Shun company to cut after product.

Carrier segments after glcG-targetron fragment after enzyme is cut is cut with enzyme is used T4DNA ligase enzyme to be connected, and this ligation is carried out 10hr in 16 ℃ of water-baths, by the connection product of acquisition with CaCl ₂heat-shocked method transforms bacillus coli DH 5 alpha competent cell: 42 ℃ of thermal shock 90sec, then add 4 ℃ of LB liquid nutrient medium recovery 1hr, then by cell with the centrifugal 5min of 4500rpm, be applied on the LB solid medium flat board that contains 100 μ g/mL penbritins and cultivate 16-18hr.

The bacterium colony obtaining is carried out to bacterium colony PCR, and (reaction reagent is by the Targetron of Sigma-Aldrich ^tMgene Knockout System (TA0100) test kit provides, condition: 95 ℃ of 5min, 94 ℃ of 30s, 55 ℃ of 30s, 30 circulations of 72 ℃ of 30s, 72 ℃ of 2min, 4 ℃ of preservations), whether to detect the targetron fragment of 350bp, connect in pWJ1 carrier, pcr amplification primer is IBS and EBS1d.

The demonstration of PCR detected result, bacterium colony PCR can amplify 350bp specific band.The bacterium colony that picking PCR is positive immediately spreads cultivation with LB liquid nutrient medium, extracts plasmid.Then,, as primer, the plasmid of extraction checks order as template to using dpIMP1-up (SEQ ID NO:7), result as expected: targetron fragment has really connected into pWJ1 carrier).

the structure of embodiment 2. clostridium acetobutylicum glcG mutant strains, detect and the loss that knocks out plasmid

By pWJ1-glcG plasmid through intestinal bacteria ER2275/pANS1 after Cac8I site methylates, electricity turns clostridium acetobutylicum ATCC 824, after recovery is spent the night, getting 200 μ l enchylema coats on the CGM flat board that is added with 20 μ g/mL erythromycin, in anaerobic box, cultivate after 48-96 hour for 37 ℃, picking list bacterium carries out bacterium colony PCR checking, and detailed process is as follows:

Methylating of 2.1pWJ1-glcG plasmid

For preventing being degraded by its restriction system cutting after foreign DNA from entering clostridium acetobutylicum, need be to the pWJ1-glcG plasmid (Mermelstein that methylates, L.D and Papoutsakis, E.T.Appl Environ Microbiol.vol 59.issue 4:p1077-81).

By pANS1 plasmid with CaCl ₂heat-shocked method is transformed into intestinal bacteria ER2275, obtains bacterial strain intestinal bacteria ER2275/pANS1.

The pWJ1-glcG plasmid that extracting is obtained is transformed into intestinal bacteria ER2275/pANS 1 competent cell, because pANS1 plasmid has spectinomycin resistance, therefore coat on the LB culture medium flat plate that contains 100 μ g/mL penbritins and 50 μ g/mL spectinomycins after overnight incubation, picking list bacterium colony is connected to incubated overnight in the LB liquid nutrient medium that 4mL is added with 100 μ g/mL penbritins and 50 μ g/mL spectinomycins, obtain the intestinal bacteria ER2275 containing pANS1 and pWJ1-glcG, with plasmid extraction test kit extracting plasmid, the plasmid that extracting is obtained is used SatI enzyme to cut checking, and (pSY6-ccpA of the unconverted intestinal bacteria ER2275/pANS1 of entering of take is contrast, satI is the isoschizomers of Cac824I, there is identical recognition site with Cac824I), enzyme is cut result and is shown, plasmid pWJ1-glcG through above-mentioned processing can not be cut by SatI enzyme, and contrast can be cut by SatI enzyme, according to enzyme, cut result, through the Cac824I restriction enzyme site of the plasmid pWJ1-glcG of above-mentioned processing, methylated and by the restriction system of clostridium acetobutylicum, do not identified.

2.2pWJ1-glcG plasmid electricity proceeds to clostridium acetobutylicum ATCC 824

Clostridium acetobutylicum ATCC 824 after streak culture 48hr, is cultivated to 16hr in picking list bacterium colony access 5mL CGM liquid nutrient medium on CGM culture medium flat plate, then by cultivating in 1% inoculum size access 50mL CGM liquid nutrient medium, when cultivating the OD of thalline ₆₀₀while reaching between 0.6-0.7, take out and cultivate bacterium, for the preparation of electricity, turn competent cell.Get 30mL bacterium liquid, in 4 ℃, the centrifugal 10min of 4500rpm, abandon supernatant, add the ETM damping fluid of 30mL4 ℃ to suspend, then in 4 ℃, the centrifugal 10min of 4500rpm, abandon supernatant, add the ET damping fluid of 4 ℃ of 1mL, obtain suspension bacteria liquid.

Get above-mentioned suspension bacteria liquid 190 μ L, add 10 μ L (approximately 1 ~ 3 μ g) the pWJ1-glcG plasmid (on ice operation) that methylates, after mixing, proceed to (2mm diameter) in electric revolving cup, use Bio-Rad MicroPulser ^tMelectroporation electricity turns, voltage 1.8kV, and all the other are with reference to service manual, the CGM substratum 1mL that adds rapidly normal temperature after electric shock, after 37 ℃ of cultivation 8hr, get 200 μ L enchylema and coat on the CGM flat board that is added with 20 μ g/mL erythromycin, in anaerobic box, cultivate approximately 2～4 days for 37 ℃.

The PCR checking of 2.3 bacterium colonies

After pWJ1-glcG plasmid is transformed in clostridium acetobutylicum ATCC 824, the partial sequence of two class introns may be inserted in genomic glcG gene, whether there is Intron insertion can use the primer of insertion point upstream and downstream, by bacterium colony PCR, verified that (wild-type bacteria that does not insert intron will amplify the band of 400bp, the recombinant bacterial strain that is inserted with intron is 1.3Kb band by the band amplifying), therefore, random five transformants of picking are verified, wherein, with the negative contrast of clostridium acetobutylicum ATCC 824 genome, detailed process is as follows:

The primer that PCR reaction is used is glcG_126-145 and glcG_473-492, and its sequence is respectively as shown in SEQID No.:8 and SEQ ID No.:9;

PCR reaction system: identical with embodiment 1;

PCR reaction conditions: 95 ℃ of 5min; 95 ℃ of 30s, 55 ℃ of 30s, 72 ℃ of 1.5min, 30 circulations; 72 ℃ of 5min.

The product that PCR reaction is obtained, carries out agarose gel electrophoresis detection, and result as shown in Figure 2.

According to the result of Fig. 2, five transformants that obtain are the mutant that has inserted intron.

2.4 sequence verification positive transformants

In random picking step 2.3 corresponding to the positive transformant that is labeled as 4 in Fig. 2, with after being added with the CGM liquid nutrient medium of 20 μ g/mL erythromycin and cultivating, extracting genome.The genome of extracting of take is template, take glcG-_126-145 and glcG_473-492 as primer pair carries out pcr amplification, reclaims 1.3kb DNA band order-checking that amplification obtains, and result is as shown in SEQ ID NO.:10.Sequencing result shows, the intron sequences of the DNA in the 101-1015 site of this sequence for inserting, and intron sequences is accurately inserted between the 269|270 site of expectation.

2.5824/pWJ1-glcG knock out the loss of plasmid

In the test tube that the transformant that 10 μ l are grown to logarithmic phase is switched to 5ml CGM nonreactive and contains erythromycin (20 μ g/ml), once, until resistance test tube is no longer grown, this process needs approximately 2 days in switching in 12～15 hours.During by the not regrowth of resistance test tube correspondence for time nonreactive test tube bacterium liquid coated plate, bacterium colony PCR, sequence verification (with 2.3,2.4) guarantee the insertion of intron, the mutant strain 824glcG that loss is knocked out to plasmid is for follow-up metabolic engineering.

the fermentation of embodiment 3.824glcG mutant strain

Get interruption in step 2.5 the clostridium acetobutylicum bacterial strain 824glcG of glcG gene in P2 substratum, ferment, and detect fermented liquid, detailed process is as follows:

From CGM flat board, in picking list bacterium access 5mL CGM liquid nutrient medium, incubated overnight, in 1% inoculum size access 50mL CGM substratum, cultivates 8 ~ 10hr, makes the dense OD of bacterium ₆₀₀reach 0.4, with cultivation and fermentation in 5% access P2 substratum, getting fermented liquid detection residual sugar content (uses the sugar-park post of WATERS company to measure through Agela 1200HPLC, result as shown in Figure 1), and with clostridium acetobutylicum ATCC 824 in contrast, wherein, before the residual sugar content in mensuration fermented liquid, need to carry out following pre-treatment: fermented liquid is after centrifugal, get respectively supernatant liquor, with H ₂o after 20 times of dilutions for residual glucose.

Result shows: glcG gene is after inserting inactivation, and under glucose exists, the final consumption of pectinose and wood sugar has increased respectively by 94% and 117% (Fig. 1).This result has proved that glcG has significantly weakened glucose retarding effect after inserting inactivation.Fig. 1 shows that result shows that wild type strain has consumed 9.87g/L pectinose in 40g/L glucose and the fermentation of 23g/L pectinose muscovado, and glcG deactivated strain has consumed 19.15g/L pectinose; In 41g/L glucose and the fermentation of 22g/L wood sugar muscovado, wild type strain has consumed 5.62g/L wood sugar, and glcG deactivated strain has consumed 12.17g/L wood sugar.Wild type strain has finally consumed the wood sugar that accounts for total reducing sugar amount approximately 8.8%.

Although knock out glcG in this research, earlier fermentation glucose utilization speed is decreased, but the fermentation period of glucose does not postpone (Figure 1A and B), not to sacrifice the utilization that is used to exchange for the secondary carbon sources such as wood sugar, pectinose of glucose.

embodiment 4.824glcG be take different times glucokinase enzyme activity in the P2 fermentation that 4%w/v glucose is carbon source mensuration

From CGM flat board, in picking list bacterium access 5mL CGM liquid nutrient medium, incubated overnight, in 1% inoculum size access 50mL CGM substratum, cultivates 8 ~ 10hr, makes the dense OD of bacterium ₆₀₀reach 0.4, cultivation and fermentation in access 950mlP2 substratum, and with clostridium acetobutylicum ATCC 824 in contrast, work as OD ₆₀₀=1.8 and during 4-4.5,4 ℃, 5000rpm, the centrifugal collection of 10min 250ml thalline is also used liquid nitrogen flash freezer.Cell after quick-frozen is resuspended in to the Tris-HCl damping fluid (50mM that 6ml contains 10%v/v glycerine, pH7.4) in, with One Shot cell crushing instrument (Low March, Daventry, Northants, UK) by broken (30KPSI, 2 times) after, 4 ℃, 12000rpm, the centrifugal collection supernatant of 30min is for enzyme activity determination.Enzyme activity determination method referring to document (Seno, E.T. and K.F.Chater (1983). journal ofGeneral Microbiology129 (May): utilize enzyme coupled reaction to detect the method that NADP reduces 1403-1413.).

Table 2 be depicted as 824 and 824glcG producing acid phase (OD ₆₀₀=1.8) and produce solvent phase (OD ₆₀₀=4-4.5) the ratio vigor of glucokinase in 4% glucose P2 substratum.

The ratio vigor of the glucokinase of table 2.824 and 824glcG

This result shows: the vigor of the glucokinase of 824glcG exceeds 0.9,3.6 times than wild-type respectively in product acid phase and product solvent phase.The rapid raising that this result has confirmed to produce solvent phase glucokinase enzyme activity has well made up produces the phenotype that acid phase glucose utilization lags behind, and the fine time of having explained that mutant strain is finished glucose not have the reason of hysteresis yet.That is the reason that, in embodiment 3, glucose utilization does not have to postpone is glucose transport and the follow-up phosphorylation that the activation of the non-PTS system of glucose has compensated mutant strain 824glcG.

The above results shows: glcG gene can become the important transformation target spot of eliminating clostridium acetobutylicum glucose retarding effect.

embodiment 5.824 bacterial strains are in the wood sugar of 1.5%w/v and the fermentation in pectinose

From CGM flat board, in picking list bacterium access 5mL CGM liquid nutrient medium, incubated overnight, in 1% inoculum size access 50mL CGM substratum, cultivates 8 ~ 10hr, makes the dense OD of bacterium ₆₀₀reach 0.4, with cultivation and fermentation in 5% access 100ml P2 substratum, getting fermented liquid detection residual sugar content (uses the sugar-park post of WATERS company to measure through Agela 1200HPLC, result as shown in Figure 7), before wherein measuring the residual sugar content in fermented liquid, need to carry out following pre-treatment: fermented liquid is after centrifugal, get respectively supernatant liquor, with H ₂o after 20 times of dilutions for residual glucose.

Result shows: the pectinose that 824 bacterial strains just can completely consumed 17.16g/L in 40 hours, and xylose utilization at this moment has just just started, and still has the residual of 1.41g/L wood sugar after 80 hours.Low sugar fermentation energy avoids too much end product as the toxic action of the thalline such as butanols, and 824 bacterial strains still show and utilize incomplete phenotype in low wood-sugar fermentation, and the natural existing problems of 824 xylose metabolism approach are described except CCR effect, product suppress.Therefore, need to seek a kind of novel method of improving this pathways metabolism.

embodiment 6. structure pIMP1-thl-xylT, pIMP1-thl-xylA, pIMP1-thl-xylB, pIMP1-thl-xylBA, pIMP1-thl-xylT – thl and pIMP1-thl-xylT-thl-xylBA plasmid vector

Clostridium acetobutylicum ATCC 824 genomes of take are template, design primer amplification xylT, thl primer, ylB and xylA, and enzyme is cut after processing the carrier of cutting processing with same enzyme and is connected, transforms DH5 α, and bacterium colony PCR checking extracting plasmid check order.Wherein, conversion, colony polymerase chain reaction (PCR) method are cut, connected to PCR, enzyme with embodiment 1.Detailed process is as follows:

The structure of 4.1pIMP1-thl-xylT

Take xylT-up and xylT-dn goes out xylT fragment as primer amplification, and its sequence is respectively as shown in SEQ ID No.:20 and SEQ ID No.:17.Use SalI and XbaI respectively enzyme cut carrier pIMP1-thl and xylT fragment, and the two identifies there is the bacterium colony extracting plasmid of positive band with same primer after connecting, transform DH5 α, the correct rear bacterium that protects of sequence verification.

The structure of 4.2pIMP1-thl-xylA

Take xylA-up and xylA-dn goes out xylA fragment as primer amplification, and its sequence is respectively as shown in SEQ ID No.:21 and SEQ ID No.:22.Use BamHI and SmaI respectively enzyme cut carrier pIMP1-thl and xylA fragment, and the two identifies there is the bacterium colony extracting plasmid of positive band with same primer after connecting, transform DH5 α, the correct rear bacterium that protects of sequence verification.

The structure of 4.3pIMP1-thl-xylB

Take xylB-up and xylB-dn goes out xylB fragment as primer amplification, and its sequence is respectively as shown in SEQ ID No.:23 and SEQ ID No.:24.Use BamHI and EcoRI respectively enzyme cut carrier pIMP1-thl and xylB fragment, and the two identifies there is the bacterium colony extracting plasmid of positive band with same primer after connecting, transform DH5 α, the correct rear bacterium that protects of sequence verification.

The structure of 4.4pIMP1-thl-xylBA

Take xylB-up and xylA-dn goes out xylBA fragment as primer amplification, and its sequence is respectively as shown in SEQ ID No.:23 and SEQ ID No.:22.Use BamHI and SmaI respectively enzyme cut carrier pIMP1-thl and xylBA fragment, and the two identifies there is the bacterium colony extracting plasmid of positive band with same primer after connecting, transform DH5 α, the correct rear bacterium that protects of sequence verification.

The structure of 4.5pIMP1-thl-xylT-thl

Take Thl2-up and Thl2-dn goes out thl promoter fragment as primer amplification, and its sequence is respectively as shown in SEQ IDNo.:25 and SEQ ID No.:26.Use XbaI and BamHI respectively enzyme cut carrier pIMP1-thl-xylT and thl promoter fragment, the two connect, transform after DH5 α with primer dxylT-overlapping-up and dpIMP 1-dn identify, its sequence is respectively as shown in SEQ ID No.:18 and SEQ ID No.:14.To there is the bacterium colony extracting plasmid of positive band, after sequence verification is correct, protect bacterium.

The structure of 4.6pIMP 1-thl-xylT-thl-xylBA

Take xylBA-up and xylBA-dn goes out xylBA operon as primer amplification, and its sequence is respectively as shown in SEQ IDNo.:27 and SEQ ID No.:28.Use BamHI and SmaI respectively enzyme cut carrier pIMP1-thl-xylT-thl and xylBA operon, the two connect, transform after DH5 α with primer dxylT-overlapping-up and dxylBA-overlapping-dn identifies, its sequence is respectively as shown in SEQ ID No.:18 and SEQ ID No.:19.To there is the bacterium colony extracting plasmid of positive band, after sequence verification is correct, protect bacterium.

embodiment 7. clostridium acetobutylicums 824 (pIMP1-thl-xylBA), 824 (pIMP1-thl-xylT), 824glcG (pIMP1-thl), 824glcG (pIMP1-thl-xylT), 824glcG (pIMP1-thl-xylA), the structure of 824glcG (pIMP1-thl-xylB) and 824glcG (pIMP1-thl-xylT-thl-xylBA) mutant strain with detection

Electricity after building correct pIMP1-thl-xylBA, pIMP1-thl-xylT in embodiment 6 and methylating is turned and enters ATCC824; Electricity after building correct pIMP1-thl-xylT, pIMP1-thl-xylA, pIMP1-thl-xylB, pIMP1-thl-xylT-thl-xylBA in pIMP 1-thl and embodiment 6 and methylating is turned and enters 824glcG, and detailed process is as follows:

Methylating of 5.1 each plasmids

Method is with 2.1

The electricity of 5.2 methylated each plasmids turns

Method is with 2.2

The bacterium colony PCR checking of 5.3 each engineering bacterias

PCR system, method, the checking of DNA agarose electrophoresis are with 2.3, and positive control is for to build correct plasmid separately, and negative control is water.

5.3.1 the evaluation of clostridium acetobutylicum 824 (pIMP1-thl-xylT)

Primer is dxylT-up and dpIMP1-dn, and its sequence is respectively as shown in SEQ ID No.:13 and SEQ ID No.:14; The positive bacterium colony obtaining is referred to as 824-xylT.

5.3.2 the evaluation of clostridium acetobutylicum 824 (pIMP1-thl-xylBA)

Primer is dpIMP1-up and dxylB-dn, and its sequence is respectively as shown in SEQ ID No.:11 and SEQ ID No.:16; The positive bacterium colony obtaining is referred to as 824-xylBA.

5.3.3 the evaluation of clostridium acetobutylicum 824glcG (pIMP1-thl)

Primer is dpIMP1-up and dthl-dn, and its sequence is respectively as shown in SEQ ID No.:11 and SEQ ID No.:12; The positive bacterium colony obtaining is referred to as 824glcG-thl.

5.3.4 the evaluation of clostridium acetobutylicum glcG (pIMP1-thl-xylT)

Primer is dxylT-up and dpIMP1-dn, and its sequence is respectively as shown in SEQ ID No.:13 and SEQ ID No.:14; The positive bacterium colony obtaining is referred to as 824glcG-xylT.

5.3.5 the evaluation of clostridium acetobutylicum glcG (pIMP1-thl-xylA)

Primer is dxylA-up and dpIMP1-dn, and its sequence is respectively as shown in SEQ ID No.:15 and SEQ ID No.:14; The positive bacterium colony obtaining is referred to as 824glcG-xylA.

5.3.6 the evaluation of clostridium acetobutylicum glcG (pIMP1-thl-xylB)

Primer is dpIMP1-up and dxylB-dn, and its sequence is respectively as shown in SEQ ID No.:11 and SEQ ID No.:16; The positive bacterium colony obtaining is referred to as 824glcG-xylB.

5.3.7 the evaluation of clostridium acetobutylicum glcG (pIMP1-thl-xylT-thl-xylBA)

First pair of primers designed is dpIMP1-up and xylT-dn, and its sequence is respectively as shown in SEQ ID No.:11 and SEQ ID No.:17, and qualification result is shown in that Fig. 4 take all swimming lanes that A is mark; The second pair of primers designed be dxylT-overlapping-up and dxylBA-overlapping-dn, its sequence is respectively as shown in SEQ ID No.:18 and SEQ ID No.:19, qualification result is shown in that Fig. 4 take all swimming lanes that B is mark.From electrophoretogram: the bacterium colony that is labeled as No. 6 is positive.The positive bacterium colony obtaining is referred to as 824glcG-TBA.

the P2 fermentation that embodiment 8.824-xylT is carbon source at 4%w/v glucose and 2%w/v wood sugar

From CGM flat board, the access of picking list bacterium contains in 5mL CGM liquid nutrient medium, and incubated overnight, in 1% inoculum size access 50mL CGM substratum, is cultivated 8 ~ 10hr, makes the dense OD of bacterium ₆₀₀reach 0.4, with cultivation and fermentation in 5% access 100ml P2 substratum, getting fermented liquid detection residual sugar content (uses the sugar-park post of WATERS company to measure through Agela 1200HPLC, result as shown in figure 10), before wherein measuring the residual sugar content in fermented liquid, need to carry out following pre-treatment: fermented liquid is after centrifugal, get respectively supernatant liquor, with H ₂o after 20 times of dilutions for residual glucose.

Result shows: with the insensitive promotor thl of glucose, carry xylT and cross and express in wild-type, can increase the utilization of wood sugar in muscovado, 96 hours 824-xylT of fermentation termination can utilize 43% wood sugar, than the multiplex 8.02g wood sugar of wild-type.

the P2 fermentation that embodiment 9.824-xylBA is carbon source at 4%w/v glucose and 2%w/v wood sugar

From CGM flat board, the access of picking list bacterium contains in 5mL CGM liquid nutrient medium, and incubated overnight, in 1% inoculum size access 50mL CGM substratum, is cultivated 8 ~ 10hr, makes the dense OD of bacterium ₆₀₀reach 0.4, with cultivation and fermentation in 5% access 100ml P2 substratum, getting fermented liquid detection residual sugar content (uses the sugar-park post of WATERS company to measure through Agela 1200 HPLC, result as shown in Figure 9), before wherein measuring the residual sugar content in fermented liquid, need to carry out following pre-treatment: fermented liquid is after centrifugal, get respectively supernatant liquor, with H ₂o after 20 times of dilutions for residual glucose.

Result shows: with the insensitive promotor thl of glucose, carry xylBA and cross and express in wild-type, can increase the utilization of wood sugar in muscovado, 96 hours 824-xylBA of fermentation termination can utilize 38% wood sugar, than the multiplex 5.2g wood sugar of wild-type.

in embodiment 10. quantitative PCR detection 824glcG-thl and 824glcG-TBA, xylT, xylA and xylB's transcribes

In the 5mLCGM liquid nutrient medium that the access of picking list bacterium contains 25 μ g/ml erythromycin from CGM flat board, incubated overnight is to OD ₆₀₀=0.8 ~ 1.0, in 1% inoculum size access 50mL CGM substratum, cultivate 8 ~ 10hr, make the dense OD of bacterium ₆₀₀reach 0.4, cultivation and fermentation in access 950ml P2 (take 4%w/v glucose and 2%w/v wood sugar are carbon source) substratum, and with 824glcG-thl in contrast, work as OD ₆₀₀=3.8 and 7 o'clock, 4 ℃, 5000rpm, the centrifugal collection of 10min 250ml thalline is also used liquid nitrogen flash freezer.The extraction of cell RNA and the same document of the preparation of cDNA (Y.Gu, waits (2010) for Ren, C.. metab Eng12 (5): 446-454.).

Every 20 μ l real-time PCR reactions systems comprise: 10 μ l iQ SYBR Green Supermix (Bio-Rad), 200nM primer, 1 μ lcDNA template.PCR in real time is carried out in PCR in real time detector (Bio-Rad), and PCR program is: 95 ℃ of 3min; 95 ℃ of 20s, 55 ℃ of 20s, 72 ℃ of 20s, 40 circulations; 65-95 ° of C carries out solubility curve analysis.All samples has all carried out three parallel laboratory tests, averages and analyzes.In order to calculate relative expression's level, cDNA has diluted 200 times and has analyzed, referring to document (Pfaffl, M.W. (2001). nucleic Acids Res29 (9): e45.).Adopt CA_C2679 and CA_C3141 as reference gene.The primer of PCR in real time is as shown in SEQ ID NOs:29-38.

Table 3.824glcG-thl and 824glcG-TBA are at 4% glucose: 2% wood sugar is producing acid phase (OD ₆₀₀=3.8) and produce solvent phase (OD ₆₀₀=7) situation of transcribing of xylT, xylA and xylB in (take 824glcG-th1 genetic expression as 1 being benchmark).

Result shows: 824glcG-thl compares with contrast, and xylT, xylA and xylB produce the acid phase in 824glcG-TBA, product solvent delayed early transcription has obvious rise, expression strategy success.

embodiment 11. engineering bacteria clostridium acetobutylicum 824glcG, 824glcG (pIMP1-thl), 824glcG (pIMP1-thl-xylT), 824glcG (pIMP1-thl-xylA), 824glcG (pIMP1-thl-xylB) and the fermentation of 824glcG (pIMP1-thl-xylT-thl-xylBA)

Fermentation process is with embodiment 3.

As seen from Figure 3: although compare with 824glcG, empty plasmid imports 824glcG makes thalline to the utilization of wood sugar, reduce by 32% in mixing sugar, but xylT, xylA, these three kinds of genes of xylB import respectively or simultaneously 824glcG makes thalline to the utilization of wood sugar, have raising in various degree in mixing sugar: compare with contrasting 824glcG-thl bacterial strain, their xylose utilization rate has improved respectively 75%, 106%, 63% and 137%.In all engineering bacterias, the engineering bacteria 824glcG-TBA that three genes import 824glcG simultaneously has the highest wood sugar rate of consumption, is the engineering bacteria that has application prospect most.

This result has proved to cross expresses one or more in xylT, xylA, xylB gene, can promote the utilization ratio of clostridium acetobutylicum to wood sugar.

embodiment 12. engineering bacteria clostridium acetobutylicum 824glcG (pIMP1-thl-xylT-thl-xylBA) are at 5.5%w/v fermentation in emulation feed liquid

From CGM flat board, in picking list bacterium access 5mL CGM liquid nutrient medium, incubated overnight, in 3% inoculum size access 100mL CGM substratum, cultivates 4～8hr, the dense OD of bacterium ₆₀₀reach 0.8～1.0, cultivation and fermentation in access 1.4LP2 substratum, getting fermented liquid detection residual sugar content (uses the sugar-park post of WATERS company to measure through Agela 1200HPLC, result is as shown in Figure 5) and acetone, butanols and ethanol content (use Agela 7890A gas chromatograph for determination, result as shown in Figure 6), with clostridium acetobutylicum ATCC 824 in contrast, wherein, before the residual sugar content in mensuration fermented liquid and acetone, butanols and ethanol content, need to carry out following pre-treatment:

Fermented liquid is got respectively supernatant liquor and is measured residual SA, butanols, ethanol after centrifugal:

Supernatant liquor is with H ₂o after 20 times of dilutions for residual glucose; Getting in 400 μ L supernatant liquors and 100 μ L mark mixes and measures acetone, butanols and ethanol (interior standard configuration side is: 25g isopropylcarbinol, 5g isopropylformic acid, 50mL 37% concentrated hydrochloric acid, adds water and be settled to 1L).

According to the result of Fig. 5, engineering bacteria 824glcG-TBA was finished the various sugar in fermented liquid substantially at 52 hours, and wild-type also has 51% wood sugar residual to fermentation termination (71h).

According to the result of Fig. 6, the butanols in 824glcG-TBA mutant strain fermented liquid and the final concentration of ABE are all higher than wild strain.

Table 4 is depicted as 3.8% glucose: 1.4% wood sugar: 0.3% pectinose fermentation 824 and residual sugar, acetone, butanols, ethanol production and the productivity of 824glcG-TBA, the calculation result of yield.

The remaining sugar concentration of table 4.824 and 824glcG-TBA bacterial strain, product output and productivity, yield

According to the result of table 4, the productivity of 824glcG-TBA mutant strain, yield are all higher than wild strain.

the fermentation of embodiment 13. clostridium acetobutylicum EA2018,2018glcG and 2018glcG-TBA

Except based on EA2018 rather than ATCC824 bacterial strain, the structure of engineering bacteria 2018glcG and 2018glcG-TBA is identical with 824glcG, 824glcG-TBA with detection.Fermentation is with embodiment 3.

Result shows: EA2018 compares with wild-type, and 2018glcG, 2018glcG-TBA have improved respectively 1.1,2.2 times the xylose utilization rate of 96 hours, and the trend of its raising is similar to the transformation result (Figure 11) in ATCC 824; Compare with wild-type, 2018glcG, 2018glcG-TBA have improved respectively 46%, 55% (Figure 12) at the ABE of 96 hours.

In sum, in method provided by the invention, xylT, xylB and xylA gene overexpression, the bacterial strain of glcG gene after two class Intron insertion inactivations utilize the ability of wood sugar and pectinose in mixing sugar to significantly improve, the corresponding raising of ABE concentration simultaneously transform generating, therefore has and utilizes ligno-cellulose hydrolysate to carry out the potentiality of acetone butanol fermentation through the bacterial strain of through engineering approaches.

All documents of mentioning in the present invention are all quoted as a reference in this application, just as each piece of document, are quoted as a reference separately.In addition should be understood that those skilled in the art can make various changes or modifications the present invention after having read above-mentioned teachings of the present invention, these equivalent form of values fall within the application's appended claims limited range equally.

Claims

1. a genetically engineered clostridium acetobutylicum, it is compared with wild-type clostridium acetobutylicum, and glcG genetic expression is suppressed or glcG protein vigor is suppressed, and the sequence of described glcG gene is CA_C (0570).

2. genetically engineered clostridium acetobutylicum as claimed in claim 1, it also has one or more features that are selected from lower group: by the wood sugar translocator of xylT genes encoding, crossed and expressed or vigor improves, by the xylose isomerase of xylA genes encoding, crossed and expressed or vigor improves and/or crossed and expressed or vigor improves by the xylulokinase of xylB genes encoding, described xylT gene is selected from: CA_C1345, CEA_G1359, described xylB gene is selected from: CA_C2612, CEA_G2621, described xylA gene is selected from: CA_C2610, CEA_G2619.

3. genetically engineered clostridium acetobutylicum as claimed in claim 1 or 2, it is characterized in that, by being selected from, one or more the genetically engineered processing of lower group obtains described genetically engineered clostridium acetobutylicum: in glcG gene, insert DNA fragmentation, partly or entirely knock out glcG gene, introduce for the antisense nucleic acid of glcG gene or interfere RNA, introducing glcG inhibition.

4. genetically engineered clostridium acetobutylicum as claimed in claim 2, is characterized in that, described genetically engineered processing comprises: import extra xylT, xylA and/or xylB gene; Introduce and improve xylT, xylA and/or the coded expression of albumen or the sudden change of vigor of xylB; Or provide the expression vector of transient expression xylT, xylA and/or the coded albumen of xylB.

5. genetically engineered clostridium acetobutylicum as claimed in claim 2, is characterized in that,

Described wood sugar translocator is albumen or its bioactive fragment for wood sugar transhipment that comes from clostridium acetobutylicum;

Described xylose isomerase is enzyme or its bioactive fragment for catalysis wood sugar generation isomery that comes from clostridium acetobutylicum;

Described xylulokinase is the enzyme for catalysis xylulose phosphate or its bioactive fragment that comes from clostridium acetobutylicum.

6. genetically engineered clostridium acetobutylicum as claimed in claim 1, is characterized in that, female bacterium of described genetically engineered clostridium acetobutylicum is selected from: ATCC 824, EA 2018.

7. genetically engineered clostridium acetobutylicum as claimed in claim 6, it is characterized in that, described genetically engineered clostridium acetobutylicum also comprises one or more plasmids that are selected from lower group: pIMP1-th1-xylT, pIMP1-th1-xylA, pIMP1-th1-xylB, pIMP1-th1-xylT-th1-xylBA, pIMP1-th1-xylT-th1-xylA, pIMP1-th1-xylT-th1-xylB, pIMP1-th1-xylBA or the promotor th1 substituting in above-mentioned plasmid with ptb or adc promotor builds each corresponding plasmid.

8. produce a method for acetone, butanols and/or ethanol, described method comprises step:

A) in suitable substratum, cultivate the bacterial strain as described in any one in claim 1-7;

B) separated product acetone, butanols and/or ethanol from culture a).

9. improve the method for clostridium acetobutylicum to the utilization ratio of wood sugar and/or pectinose, described method comprises step:

(a) clostridium acetobutylicum is carried out to genetically engineered transformation, to suppress glcG genetic expression for wild-type clostridium acetobutylicum, the sequence of described glcG gene is CA_C (0570);

10. method as claimed in claim 9, it is characterized in that, step (a) also comprises carries out genetically engineered transformation to described clostridium acetobutylicum, with for wild-type clostridium acetobutylicum: the expression or the vigor that improve the wood sugar translocator of being encoded by xylT, the expression of the xylose isomerase that raising is encoded by xylA or vigor, and/or improve expression or the vigor of the xylulokinase encoded by xylB, described xylT gene is selected from: CA_C1345, CEA_G1359, described xylB gene is selected from: CA_C2612, CEA_G2621, described xylA gene is selected from: CA_C2610, CEA_G2619.

11. 1 kinds are improved the method that clostridium acetobutylicum is produced the productive rate of product acetone, butanols and/or ethanol, and described method comprises step:

12. methods as claimed in claim 11, it is characterized in that, step (a) also comprises carries out genetically engineered transformation to described clostridium acetobutylicum, with for wild-type clostridium acetobutylicum: improve by the expression of the wood sugar translocator of xylT genes encoding or vigor, raising by expression or the vigor of the xylose isomerase of xylA genes encoding and/or improve expression or the vigor by the xylulokinase of xylB genes encoding, described xylT gene is selected from: CA_C1345, CEA_G1359; Described xylB gene is selected from: CA_C2612, CEA_G2621; Described xylA gene is selected from: CA_C2610, CEA_G2619.

13. methods as described in any one in claim 9-12, is characterized in that,

Described inhibition glcG genetic expression is to realize by being selected from one or more modes of lower group: in glcG gene, insert DNA fragmentation, partly or entirely knock out glcG gene, introduce antisense nucleic acid or interfere RNA, introducing glcG inhibition.

14. methods as described in claim 10 or 12, is characterized in that, described genetically engineered transformation comprises: import extra xylT, xylA and/or xylB gene; Introduce and improve xylT, xylA and/or the coded expression of albumen or the sudden change of vigor of xylB; Or the expression vector of transient expression xylT, xylA and/or the coded albumen of xylB is provided, described xylT gene is selected from: CA_C1345, CEA_G1359; Described xylB gene is selected from: CA_C2612, CEA_G2621; Described xylA gene is selected from: CA_C2610, CEA_G2619.

15. methods as described in claim 10 or 12, is characterized in that,

16. methods as described in any one in claim 9-12, is characterized in that, described clostridium acetobutylicum is selected from: ATCC 824, EA 2018.

17. methods as claimed in claim 16, it is characterized in that, described genetically engineered clostridium acetobutylicum also transforms to be selected from one or more plasmids of lower group: pIMP1-th1-xylT, pIMP1-th1-xylA, pIMP1-th1-xylB, pIMP1-th1-xylT-th1-xylBA, pIMP1-th1-xylT-th1-xylA, pIMP1-th1-xylT-th1-xylB, pIMP1-th1-xylBA or the promotor th1 substituting in above-mentioned plasmid with ptb or adc promotor builds each corresponding plasmid.

18. methods as described in any one in claim 9-12, is characterized in that, described in comprise wood sugar and/or pectinose raw material be selected from: the hydrolyzed solution of Mierocrystalline cellulose or hemicellulose, grain, cotton.

The purposes of 19. methods as described in any one in claim 9-18 in the production of butanols, acetone and/or ethanol.